The cyclopentenone prostaglandin A 1 (PGA 1 ) is an inducer of cell death in cancer cells. However, the mechanism that initiates this cytotoxic response remains elusive. Here we report that PGA 1 triggers apoptosis by a process that entails the specific activation of H- and N-Ras isoforms, leading to caspase activation. Cells without H- and N-Ras did not undergo apoptosis upon PGA 1 treatment; in these cells, the cellular demise was rescued by overexpression of either H-Ras or N-Ras. Consistently, the mutant H-Ras-C118S, defective for binding PGA 1 , did not produce cell death. Molecular analysis revealed a key role for the RAF-MEK-ERK signaling pathway in the apoptotic process through the induction of calpain activity and caspase-12 cleavage. We propose that PGA 1 evokes a specific physiological cell death program, through H- and N-Ras, but not K-Ras, activation at endomembranes. Our results highlight a novel mechanism that may be of potential interest for tumor treatment.

This work was supported by Grants from MEC SAF2004-02900 (PAL) and SAF 2002-03096 (AO), Fondo de Investigacion Sanitaria PI02-0585 (PAL), and Fundacion Memoria Samuel Solorzano Barruso (PAL).

Tetraspanin proteins form signaling complexes between them and with other membrane proteins and modulate cell adhesion and migration properties. The surface expression of several tetraspanin antigens (CD9, CD37, CD53, CD63, and CD81), and their interacting proteins (CD19, CD21, and HLA-DR) were analyzed during normal B-cell maturation and compared to a group of 67 B-cell neoplasias. Three patterns of tetraspanin expression were identified in normal B cells. The first corresponded to bone marrow CD10(+) B-cell precursors (BCP) which showed high expression of CD81 and CD9, low reactivity for CD53 and negativity for CD37. CD10(-) B-lymphocytes showed downregulation of CD9/CD81 and upregulation of CD53/CD37. Plasma cells showed re-expressed CD9 and downregulated CD37. Hierarchical clustering analysis of flow cytometry immunophenotypic data showed a good correlation between the tumor differentiation stage and the pattern of tetraspanin expression, with all analyzed individual samples classified into three major groups, independently of their normal or neoplastic origin. Despite this, neoplastic B-cells frequently showed aberrantly high/low expression of the different markers analyzed. Interestingly, in B-cell chronic lymphocytic leukemia, abnormal expression of CD53 and CD9 were associated with different patterns of disease infiltration, which would support the role of these molecules on modulating adhesion and migration of neoplastic B cells.

Tetraspanins are a group of hydrophobic proteins with four transmembrane domains and two extracellular loops, both with conserved residues. Some tetraspanins are cell specific and others are very ubiquitous. Tetraspanins interact with very different types of proteins such as integrins, membrane receptors, as well as intracellular signalling molecules. Tetraspanins can interact with other tetraspanins to form a larger complex, whose core is formed by six tetraspanins, surrounded several tetraspanin-associated proteins. These complexes can further aggregate and behave as a membrane microdomain. The great heterogeneity in their composition and the dynamics of tetraspanin complexes confers great flexibility on these proteins to participate in many different biological roles.

BACKGROUND:The interaction of mesangial cells with the extracellular matrix plays a major role in kidney biology. Tetraspanin proteins modulate cell interaction with the extracellular matrix. Tetraspanins form supramolecular structures on the cell membrane that send signals after engagement by unknown ligands, modulate different signaling processes, and regulate cell adhesion and motility.METHODS:CD53 was determined by immunohistochemistry, and on the cell surface of cultured rat mesangial cells by flow cytometry. Mesangial cell cultures were stimulated with MRC OX-44 antibody. DNA synthesis was measured by thymidine incorporation. Extracellular signal-regulated kinase (ERK) activation was determined by Western blot.RESULTS:CD53 was present in mesangial cells in vivo and in culture. Ligation of CD53 antigen with a monoclonal antibody triggered the induction of DNA synthesis, which was not sensitive to inhibitors of signaling pathways that use phosphatidylinositol 3-kinase (PI3K) and protein kinase C, or to calcium channel inhibitors, such as thapsigargin and verapamil. The DNA synthesis was inhibited by PD98059, a specific inhibitor of MEK that prevents ERK1/ERK2 activation. In addition, ERK1 and ERK2 activation by phosphorylation occurred following CD53 antigen ligation. The DNA synthesis was due to de novo synthesis and not to DNA repair as a consequence of the initiation of apoptosis, determined by flow cytometry, and lack of proteolytic activation of PARP by caspase 3. CD53 antigen ligation also induced an increase in mitochondrial activity.CONCLUSIONS:To our knowledge this is the first identification of a tetraspanin protein in mesangial cells. CD53 antigen delivers a signal that initiates DNA synthesis. This signal is mediated by ERK1/ERK2 activation, but it is not sufficient to complete the cell cycle.

The CD53 antigen is a member of the tetraspanin membrane protein family that is expressed in the lymphoid‐myeloid lineage. We have studied the implication of CD53 antigen in signal transduction by determining the effect of its ligation on the c‐Jun N‐terminal kinase (JNK) in different cell types. Ligation of the rat or human CD53 antigen induces a three‐ to fourfold transient activation of JNK activity that peaks at 3–5 min. The effect was detected by assaying the endogenous or exogenous (transfected) JNK activity. The JNK response was detected in IR938F cells, a rat B‐cell lymphoma, and in Jurkat cells derived from a human T‐cell lymphoma. This JNK activation was not mediated by the vav oncogene, and CD53 does not cooperate with CD3 for vav activation. A␣similar JNK activation was also detected in a human renal carcinoma cell line that was transiently transfected with the human CD53 cDNA to mimic the CD53 ectopic expression in carcinomas. In stable CD53‐transfected cells it stimulated Jun‐dependent transcriptional activity. We conclude that parts of the cell responses modulated by the CD53 are mediated by JNK activation, and this activation is independent of the different protein interactions that the CD53 protein has on specific cell types.

CD53 is a tetraspanin protein mostly expressed in to the lymphoid-myeloid lineage. We have characterized the human CD53 gene regulatory region. Within the proximal 2 kilobases, and with opposite transcriptional orientation, is located the promoter-enhancer of a second gene, which does not affect CD53. Twenty-four copies of a CA dinucleotide repeat separate these two gene promoters. The proximal enhanceosome of the human CD53 gene is comprised between residues -266 and +84, and can be subdivided into four major subregions, two of them within exon 1. Mutational analysis identified several cooperating sequences. An Sp1 and an ets-1 site, at positions -115 and +62, respectively, are essential for transcriptional competence in all cell lines. Five other regulatory sequences have a dual role, activator or down-regulator, depending on the cell line. At the end of the non-coding exon 1, +64 to +83, there is a second ets-1 regulatory element, which is required for high level of transcription, in cooperation with the Sp1 site, in K562 and Molt-4, but not in Namalwa cells, where it functions as a repressor. This Sp1 site also cooperates with another ets-1/PU.1 site at -172. Different cell types use different regulatory sequences in the enhanceosome for the expression of the same gene.

TO THE EDITORTetraspan transmembrane (TST) antigens are a family of pro-teins with unknown function which are increasingly impli-cated in cancer biology. TST proteins are defined by theirstructural characteristics. There are at least 15 known TST pro-teins in human cells, and most of them were identified astumor antigens with a heterogeneous cell type distribution.

SUMMARYTetraspanin antigens are implicated in the prognosis of different types of tumours. In this study we determine by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) the level of 13 tetraspan messages in 21 Burkitt lymphoma (BL) cell lines. All tumour cell lines have a common pattern of tetraspanin gene expression. There are five antigens which are detected in 90% of cell lines at high levels, CD53, CD81, CD63, SAS and CD82. Another two, CD9 and CD37, were detected in 60% of cell lines, and have a very variable level of expression. The remaining antigens, A15, CoO29, KRAG, L6, TI-1 and il-TMP, are expressed at low levels in very few cell lines without any specific pattern. The level of gene expression corresponds with the level of cell surface antigen determined by flow cytometry. The average number of tetraspan proteins expressed per cell line is six. These proteins may form subunits of an oligomeric structure with 24 transmembrane domains. There are no major differences in tetraspan expression pattern among sporadic or endemic tumours, type of translocation or Epstein–Barr virus status, suggesting the original cell of these tumours is the same, probably a late pre-B cell, at the CD9 to CD37 transition point. Tetraspanin gene expression is consistent with BL being a single entity, despite variations in other parameters.